<i>Escherichia coli</i>Methionine Aminopeptidase:  Implications of Crystallographic Analyses of the Native, Mutant, and Inhibited Enzymes for the Mechanism of Catalysis<sup>,</sup>

Lowther, W. Todd; Orville, Allen M.; Madden, David T.; Lim, Seung-Taek; Rich, Daniel H.; Matthews, Brian W.

doi:10.1021/bi990684r

Cited by 162 publications

(245 citation statements)

References 57 publications

Supporting

Mentioning

217

Contrasting

Unclassified

Order By: Relevance

“…It is interesting to note that during the catalysis, coordination of M1 changes from pentacoordinate to hexacoordinate after binding of the substrate. Both pentacoordinate and hexacoordinate metal environments have been seen in MetAP structures reported here and earlier with different inhibitors bound (18,20,32). Further support for the scissile carbonyl oxygen binding to M1 at the same time as a water molecule comes from a study on P. furiosus MetAP by EPR, which showed that both nucleophile and substrate bind to a catalytic Fe(II) center in that enzyme (40).…”

Section: Mono-and Dimetalated Structures Of Metap Complexed With Nlepsupporting

confidence: 73%

“…X-ray structural analyses of numerous enzymes in this family, both with and without small ligands bound at the active site, show that all possess a dinuclear metal site (dimetalated) comprising conserved amino acid residues that furnish imidazole and carboxylate donors (19). These analyses have led to proposed reaction mechanisms involving both metal ions (i.e., M1 and M2) (20,21). A water molecule (or hydroxide ion) that bridges M1 and M2 serves as a nucleophile to attack the carbonyl group of the scissile peptide bond.…”

mentioning

confidence: 99%

See 1 more Smart Citation

Structural basis of catalysis by monometalated methionine aminopeptidase

Xie

Qiang

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Methionine aminopeptidase (MetAP) removes the amino-terminal methionine residue from newly synthesized proteins, and it is a target for the development of antibacterial and anticancer agents. Available x-ray structures of MetAP, as well as other metalloaminopeptidases, show an active site containing two adjacent divalent metal ions bridged by a water molecule or hydroxide ion. The predominance of dimetalated structures leads naturally to proposed mechanisms of catalysis involving both metal ions. However, kinetic studies indicate that in many cases, only a single metal ion is required for full activity. By limiting the amount of metal ion present during crystal growth, we have now obtained a crystal structure for a complex of Escherichia coli MetAP with norleucine phosphonate, a transition-state analog, and only a single Mn(II) ion bound at the active site in the position designated M1, and three related structures of the same complex that show the transition from the mono-Mn(II) form to the di-Mn(II) form. An unliganded structure was also solved. In view of the full kinetic competence of the monometalated MetAP, the much weaker binding constant for occupancy of the M2 site compared with the M1 site, and the newly determined structures, we propose a revised mechanism of peptide bond hydrolysis by E. coli MetAP. We also suggest that the crystallization of dimetalated forms of metallohydrolases may, in some cases, be a misleading experimental artifact, and caution must be taken when structures are generated to aid in elucidation of reaction mechanisms or to support structure-aided drug design efforts.metalloprotease ͉ protein structure ͉ enzyme inhibition ͉ drug discovery ͉ metal occupancy A ll newly synthesized proteins have an amino-terminal methionine residue corresponding to the start codon AUG. In a significant number of cases, this initiating methionine residue is removed, either co-or posttranslationally, by the enzyme methionine aminopeptidase (MetAP) (1). In bacteria, this enzyme is the product of a single gene, and it is absolutely essential for bacterial survival, as demonstrated by gene deletion experiments in Escherichia coli (2) and Salmonella typhimurium (3). The single but critically essential MetAP enzyme of bacteria thus stands out as an attractive target for the design of antibacterial agents (4). Eukaryotes, on the other hand, have two distinct MetAPs, types I and II, arising from different genes (5). The human type II MetAP is a target of the antiangiogenic compounds fumagillin, ovalicin, and TNP-470 (6-8). Bengamides inhibit both types of MetAP (9) and cause inhibition of the growth of several human tumor cell lines in vitro at low-nanomolar concentrations. Therefore, human MetAPs may also serve as targets for the development of new anticancer agents. Some small molecules that inhibit MetAPs potently in vitro are known, but they lack potent antibacterial (10-12) or antiangiogenic (13) activities. One reason for this failure may be that they do not penetrate the bacterial or mammalian cells to...

show abstract

Section: Mono-and Dimetalated Structures Of Metap Complexed With Nlepsupporting

confidence: 73%

mentioning

confidence: 99%

Structural basis of catalysis by monometalated methionine aminopeptidase

Xie

Qiang

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

show abstract

“…The 3, 4, and 5 hydroxyl groups of the inhibitor coordinate the two active-site cobalt ions. As discussed below, the geometry of the interactions of the hydroxyl groups with the di-cobalt center is remarkably similar to that observed for the main chain amide and carbonyl groups of peptidic inhibitors of aminopeptidases (24).…”

Section: Fig 4 Inhibition Of Metap Enzymatic and Antiproliferative supporting

confidence: 54%

“…6B), but is very similar to that of a bestatin-derived inhibitor of Escherichia coli MetAp (24). Bestatin is a naturally occurring peptidic inhibitor of aminopeptidases (25) and has been modified to create an inhibitor specific for E. coli MetAp by altering amino acid side chains (24).…”

Section: Fig 4 Inhibition Of Metap Enzymatic and Antiproliferative mentioning

confidence: 99%

Proteomics-based Target Identification

Towbin

Bair

DeCaprio

et al. 2003

Journal of Biological Chemistry

144

View full text Add to dashboard Cite

LAF389 is a synthetic analogue of bengamides, a class of marine natural products that produce inhibitory effects on tumor growth in vitro and in vivo. A proteomicsbased approach has been used to identify signaling pathways affected by bengamides. LAF389 treatment of cells resulted in altered mobility of a subset of proteins on two-dimensional gel electrophoresis. Detailed analysis of one of the proteins, 14-3-3␥, showed that bengamide treatment resulted in retention of the amino-terminal methionine, suggesting that bengamides directly or indirectly inhibited methionine aminopeptidases (MetAps). Both known MetAps are inhibited by LAF389. Short interfering RNA suppression of MetAp2 also altered amino-terminal processing of 14-3-3␥. A high resolution structure of human MetAp2 co-crystallized with a bengamide shows that the compound binds in a manner that mimics peptide substrates. Additionally, the structure reveals that three key hydroxyl groups on the inhibitor coordinate the di-cobalt center in the enzyme active site.

show abstract

“…The geometric arrangement of these residues is practically identical in all of the MetAP x-ray structures (11,12). Several three-dimensional structures of MetAPs have been determined by x-ray diffraction methods including the structure of human MetAP-II with a covalently bound fumagillin molecule (13)(14)(15).…”

Section: Methionine Aminopeptidases (Metaps)mentioning

confidence: 99%

Protonation States of Methionine Aminopeptidase and Their Relevance for Inhibitor Binding and Catalytic Activity

Klein

Schiffmann

Folkers³

et al. 2003

Journal of Biological Chemistry

View full text Add to dashboard Cite

We have performed a computational study of different protomeric states of the methionine aminopeptidase active site using a combined quantum-mechanical/molecular mechanical simulation approach. The aim of this study was to clarify the native protonation state of the enzyme, which is needed for the development of novel irreversible inhibitors that can possibly be used as antiangiogenic and antibiotic drugs by virtual screening and other drug design methods. The results of the simulations indicated that two protonation states are possible without disturbing the overall geometry of the active site. We then verified experimentally the presence of the two protonation states by studying the substrate hydrolysis and inhibitor binding reactions at different pH values and come to the conclusion that one of the protomeric states is relevant for inhibitor binding, whereas the other is relevant for substrate hydrolysis. This result has implications for the development of other inhibitors of this class of enzymes and adds a new perspective to the pharmacological properties of the antiangiogenic drug fumagillin, which is an irreversible inhibitor of the human methionine aminopeptidase type II.Methionine aminopeptidases (MetAPs) 1 play a central role for in vivo protein synthesis as they remove the starter methionine from newly synthetized proteins. The natural product fumagillin, an epoxide, covalently modifies one of the activesite histidines in the eukaryotic methionine aminopeptidase II (MetAP-II) (1-4) and other MetAPs (cf. Fig. 1). Fumagillin inhibits the growth of vessels in tumors, and a derivative of the compound has been evaluated in clinical trials as an anticancer drug. The antiangiogenic effect of fumagillin and other inhibitors of MetAP-II have been attributed to the inhibition of the Ets-1 transcription factor expression and the activation of the p53 pathway (5, 6). Besides from being anticancer drug targets, MetAPs have the potential to become the target proteins of antibacterial substances because the MetAP functionality is essential for cell growth and bacteria possess only one of the two known MetAP subtypes (7).MetAPs are metal-dependent enzymes. It is not clear which metal activates the MetAPs in vivo. For in vitro experiments, cobalt is commonly used because it activates all known MetAPs and the cobalt-substituted enzymes are usually the most active (8). Zinc and iron(II) have also been shown to activate some MetAPs (9, 10). The metal-chelating residues in all known MetAPs are two glutamates, two aspartates, and one histidine. The geometric arrangement of these residues is practically identical in all of the MetAP x-ray structures (11,12). Several three-dimensional structures of MetAPs have been determined by x-ray diffraction methods including the structure of human MetAP-II with a covalently bound fumagillin molecule (13-15).Density functional theory (16, 17) has long been used to study the reactivity of organic molecules and recently began to find its way into biochemistry (18). We present here an ex...

show abstract

Escherichia coliMethionine Aminopeptidase: Implications of Crystallographic Analyses of the Native, Mutant, and Inhibited Enzymes for the Mechanism of Catalysis^,

Cited by 162 publications

References 57 publications

Structural basis of catalysis by monometalated methionine aminopeptidase

Structural basis of catalysis by monometalated methionine aminopeptidase

Proteomics-based Target Identification

Protonation States of Methionine Aminopeptidase and Their Relevance for Inhibitor Binding and Catalytic Activity

Contact Info

Product

Resources

About

Escherichia coliMethionine Aminopeptidase: Implications of Crystallographic Analyses of the Native, Mutant, and Inhibited Enzymes for the Mechanism of Catalysis,

Cited by 162 publications

References 57 publications

Structural basis of catalysis by monometalated methionine aminopeptidase

Structural basis of catalysis by monometalated methionine aminopeptidase

Proteomics-based Target Identification

Protonation States of Methionine Aminopeptidase and Their Relevance for Inhibitor Binding and Catalytic Activity

Contact Info

Product

Resources

About

Escherichia coliMethionine Aminopeptidase: Implications of Crystallographic Analyses of the Native, Mutant, and Inhibited Enzymes for the Mechanism of Catalysis^,